Damaged neuronal connections in the adult central nervous system (CMS) do not regenerate. This is in part due to the physical barrier formed by glial cells that respond by proliferating and forming a scar and to the presence of inhibitory molecules in the CMS environment. Several molecules have been implicated in the failure of the CMS to regenerate, but inhibiting the function of these molecules so far has produced modest improvements. Therefore, other factors must be important. Recent work has shown that the EphA4 receptor tyrosine kinase plays a critical role in the inhibition of axon regeneration that occurs after spinal cord injury. Remarkably, axons in EphA4 knockout mice can regenerate past the site of injury and re-establish severed connections resulting in functional recovery. Other evidence suggests that EphA4 plays an inhibitory role in axonal and dendritic growth in other regions of the central nervous system as well. Thus, inhibiting EphA4 function is a very promising new approach to promote regeneration in the CMS, with high potential for a number of therapeutic applications. However, EphA4 has not yet been exploited as a target for small molecules. The signaling activity of EphA4 is stimulated by binding several membrane-anchored ligands, called ephrins. As we have recently shown, peptides that antagonize ephrin binding without stimulating the signaling ability of EphA4 block the physiological activity of the receptor. In this application we propose to develop an assay system that can be used for high throughput screening of small molecule antagonists for EphA4, perform a pilot screen, and develop assays to characterize the potency and selectivity of the active compounds identified. The methodology developed will in the future allow full scale high throughput screens for small molecule EphA4 antagonists and their subsequent characterization. An advantage of reagents that target the extracellular domain of EphA4 is that they can be highly selective, unlike most kinase domain inhibitors, and that they can act without having to penetrate inside the cell. A particularly promising therapeutic application of EphA4 antagonists is in spinal cord injury, where these reagents could be applied locally to improve functional recovery.